JP2020045303A - Expression inhibitors of immune check point factors in cancer cells and pharmaceutical compositions for treating cancer - Google Patents
Expression inhibitors of immune check point factors in cancer cells and pharmaceutical compositions for treating cancer Download PDFInfo
- Publication number
- JP2020045303A JP2020045303A JP2018173611A JP2018173611A JP2020045303A JP 2020045303 A JP2020045303 A JP 2020045303A JP 2018173611 A JP2018173611 A JP 2018173611A JP 2018173611 A JP2018173611 A JP 2018173611A JP 2020045303 A JP2020045303 A JP 2020045303A
- Authority
- JP
- Japan
- Prior art keywords
- cancer
- expression
- rab3b
- immune checkpoint
- sphere
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
本発明は、癌細胞、特に癌幹細胞特性を有する癌細胞における免疫チェックポイント因子の発現抑制剤や、癌治療用医薬組成物に関する。 The present invention relates to an agent for suppressing the expression of an immune checkpoint factor in cancer cells, particularly cancer cells having cancer stem cell properties, and a pharmaceutical composition for treating cancer.
近年、自己複製能、多分化能、造腫瘍能、再発、転移能、又は治療抵抗性などを持つ癌の根本ともいえる癌幹細胞（cancer stem cells）の存在が報告されてきた。癌幹細胞の中には、薬剤耐性を獲得しているものがあるという報告もあり（非特許文献1参照）、治療によって大部分の癌細胞を除いても、ごく少数の癌幹細胞が生き残っていれば再発や転移が生じる可能性が高くなると考えられている。そのため、癌治療において、癌細胞だけでなく癌幹細胞も治療標的とすることが注目され、癌幹細胞をターゲットとする新たな癌治療の概念が報告されている。癌細胞と同時に癌幹細胞も除去することができれば、癌の再発や転移の防止にも有用な治療法の開発につながることが期待されている。 In recent years, the existence of cancer stem cells (cancer stem cells) which can be said to be the root of cancer having self-renewal ability, pluripotency, tumorigenicity, recurrence, metastasis ability, treatment resistance and the like has been reported. It has been reported that some cancer stem cells have acquired drug resistance (see Non-Patent Document 1). Even if most cancer cells are removed by treatment, only a small number of cancer stem cells can survive. It is thought that the possibility of recurrence or metastasis increases. Therefore, in cancer treatment, not only cancer cells but also cancer stem cells are attracting attention, and a new concept of cancer treatment targeting cancer stem cells has been reported. If cancer stem cells can be removed at the same time as cancer cells, it is expected to lead to the development of a therapeutic method useful for preventing cancer recurrence and metastasis.
しかしながら、癌幹細胞は癌細胞集団において極めて少数であるということだけでなく、癌細胞における不均一性(heterogeneity)と同様に、癌幹細胞においても不均一性があることが分かってきており、コンセプトに基づく癌治療法の開発は困難であった。また、癌幹細胞の研究の困難な点は、癌組織中の癌幹細胞の割合が少ないため、多くの癌幹細胞を用いた研究を行うことが難しかった。そこで本発明者らは、血清を含有しない動物細胞培養用基礎培地に、神経生存因子−１（neural survival factor-1:NSF-1）を添加した培地を用いた消化器系癌幹細胞の増殖方法（特許文献1参照）を提案した。これによって、CSCの特性を有する癌幹細胞様細胞(cancer stem-like cells:以下「CSLC」ともいう)を効率的に得ることが出来るようになった。 However, it has been found that cancer stem cells are not only a very small number in the cancer cell population, but also heterogeneous in cancer cells as well as heterogeneity in cancer cells. Development of a cancer therapy based on it has been difficult. Also, the difficulty in researching cancer stem cells is that it is difficult to conduct research using many cancer stem cells because the proportion of cancer stem cells in the cancer tissue is small. Therefore, the present inventors have proposed a method for growing a gastrointestinal cancer stem cell using a medium obtained by adding a neural survival factor-1 (NSF-1) to a serum-free basal medium for animal cell culture. (See Patent Document 1). As a result, cancer stem-like cells (hereinafter, also referred to as “CSLC”) having CSC characteristics can be efficiently obtained.
ところで、近年、癌治療において免疫チェックポイント療法（immune checkpoint therapy）が行われるようになってきた。かかる免疫チェックポイント療法は、癌の免疫監視機構を利用して癌細胞を破壊する療法である。免疫チェックポイント療法に用いられる免疫チェックポイント阻害剤としては、抗CTLA-4抗体、抗PD-1抗体、抗PD-L1抗体等が知られている。 By the way, in recent years, immune checkpoint therapy has been performed in cancer treatment. Such an immune checkpoint therapy is a therapy that uses an immune monitoring mechanism of cancer to destroy cancer cells. Known immune checkpoint inhibitors used for immune checkpoint therapy include anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody and the like.
現在免疫チェックポイント阻害剤として用いられているものは、免疫チェックポイント分子又はそのリガンドに結合して、癌の免疫逃避機構を変化させてT細胞等の細胞障害性免疫細胞の活性を制御するものが中心である。しかしながら、これらの免疫チェックポイント阻害剤のほとんどは、全身性に免疫細胞に発現している分子を標的としているため、血中サイトカインの上昇によるサイトカインストーム等の全身的な副作用が生じ、場合によっては死に至るという問題があった。そこで、本発明の課題は、癌細胞、特に癌幹細胞における免疫チェックポイント因子の発現抑制剤を提供することにある。 Currently used as immune checkpoint inhibitors are those that bind to immune checkpoint molecules or their ligands, alter the immune escape mechanism of cancer, and control the activity of cytotoxic immune cells such as T cells Is the center. However, most of these immune checkpoint inhibitors target molecules that are systemically expressed in immune cells, resulting in systemic side effects such as cytokine storms due to an increase in blood cytokines. There was a problem of death. Therefore, an object of the present invention is to provide an agent for suppressing the expression of an immune checkpoint factor in cancer cells, particularly cancer stem cells.
本発明者らは、自らが開発した独自の誘導法により、癌幹細胞の特性の一つである浮遊細胞塊（sphere）形成能を癌幹細胞の指標としてCSLCの機能的選択を行い、これによって得られた細胞群において、種々の抗癌剤に対する耐性が亢進するだけでなく、血行性肝転移能が亢進していることを免疫不全マウスにおける経門脈的肝転移モデルにおいて確認した。従来のCSCマーカーCD44v9によるソーティング法では得られる細胞数が少ないため、さらにDNA chip等の網羅的解析を行うことが困難であったが、本発明者らは癌幹細胞の性質を有する細胞を誘導することで、大量のCSLCを得ることが可能となり、RNA-seq解析(mRNA)、DNA chip解析(microRNA)を行った。さらに、癌幹細胞の性質の中でも再発・転移能に着目してヒト臨床検体でのRNA-seq解析結果も統合して解析することで、転移性肝内再発に特異的な遺伝子であるRAB3B遺伝子を同定した。 The inventors of the present invention performed functional selection of CSLC by using a proprietary induction method developed by themselves, using the ability to form a floating cell mass (sphere), which is one of the characteristics of cancer stem cells, as an indicator of cancer stem cells. In the obtained cell group, it was confirmed in a transportal liver metastasis model in immunodeficient mice that not only resistance to various anticancer drugs was enhanced but also hematogenous liver metastasis was enhanced. With the conventional sorting method using the CSC marker CD44v9, the number of cells obtained is small, so it was difficult to perform a comprehensive analysis of a DNA chip or the like, but the present inventors induce cells having the properties of cancer stem cells. Thus, a large amount of CSLC could be obtained, and RNA-seq analysis (mRNA) and DNA chip analysis (microRNA) were performed. Furthermore, focusing on recurrence and metastasis among the characteristics of cancer stem cells, the results of RNA-seq analysis on human clinical specimens are also integrated and analyzed, enabling RAB3B gene, a gene specific for metastatic intrahepatic recurrence, to be analyzed. Identified.
ここで、癌幹細胞は抗癌剤治療や放射線治療に対して抵抗性があることが知られている。また、近年では癌幹細胞の治療抵抗性に免疫療法も含まれることが示唆されている。そこで、上記sphere形成能を有するCSLCを解析したところ、複数の免疫チェックポイント関連分子の発現が変化しており、CSLCにおける免疫逃避機構が示された。さらに、CSLC特異的遺伝子であるRAB3BのCSLC免疫逃避機構への関与について解析を進める中で、癌幹細胞におけるRAB3B遺伝子をノックアウトすると免疫チェックポイント分子PD-1（programmed cell death-1）のリガンドであるPD-L1（programmed cell death-1 ligand-1）及びPD-L2（programmed cell death-1 ligand-2）の発現が抑制されることを見出し、本発明を完成した。 Here, it is known that cancer stem cells have resistance to anticancer drug treatment and radiation treatment. In recent years, it has been suggested that immunotherapy is included in the treatment resistance of cancer stem cells. Thus, when CSLC having the sphere-forming ability was analyzed, the expression of a plurality of immune checkpoint-related molecules was changed, indicating an immune escape mechanism in CSLC. Furthermore, while analyzing the involvement of the CSLC-specific gene RAB3B in the CSLC immune escape mechanism, knocking out the RAB3B gene in cancer stem cells is a ligand for the immune checkpoint molecule PD-1 (programmed cell death-1). The present inventors have found that the expression of PD-L1 (programmed cell death-1 ligand-1) and PD-L2 (programmed cell death-1 ligand-2) is suppressed, and completed the present invention.
すなわち、本発明は、以下のとおりである。
（１）以下の（I）又は（II）のいずれかを有効成分とする、癌細胞における免疫チェックポイント因子の発現抑制剤。
（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子；
（II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物；
（２）癌細胞が、癌幹細胞であることを特徴とする上記（１）記載の癌細胞における免疫チェックポイント因子の発現抑制剤。
（３）癌幹細胞が、肝癌の癌幹細胞であることを特徴とする上記（１）又は（２）記載の癌細胞における免疫チェックポイント因子の発現抑制剤。
（４）免疫チェックポイント因子が、PD-L1又はPD-L2であることを特徴とする上記（１）〜（３）のいずれか記載の癌細胞における免疫チェックポイント因子の発現抑制剤。
（５）核酸分子がsmall interfering (siRNA)、micro RNA、small hairpin RNA(shRNA)、guide RNA(gRNA)、アンチセンス核酸、又はリボザイムからなる群から選択される少なくとも１種の核酸分子であることを特徴とする上記（１）〜（４）のいずれか記載の癌細胞における免疫チェックポイント因子の発現抑制剤。
（６）核酸分子がsiRNAであることを特徴とする上記（１）〜（５）のいずれか記載の癌細胞における免疫チェックポイント因子の発現抑制剤。
（７）上記（１）〜（６）のいずれか記載の癌細胞における免疫チェックポイント因子の発現抑制剤と、薬理学的に許容される添加物を含有する癌治療用医薬組成物。
That is, the present invention is as follows.
(1) An agent for suppressing the expression of an immune checkpoint factor in cancer cells, comprising any one of the following (I) or (II) as an active ingredient:
(I) a nucleic acid molecule that targets the RAB3B gene and suppresses the expression of the RAB3B gene;
(II) a low-molecular compound that binds to the RAB3B protein represented by SEQ ID NO: 1;
(2) The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to (1), wherein the cancer cells are cancer stem cells.
(3) The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to the above (1) or (2), wherein the cancer stem cells are cancer stem cells of liver cancer.
(4) The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to any one of the above (1) to (3), wherein the immune checkpoint factor is PD-L1 or PD-L2.
(5) The nucleic acid molecule is at least one nucleic acid molecule selected from the group consisting of small interfering (siRNA), micro RNA, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, and ribozyme. The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to any one of the above (1) to (4), which is characterized in that:
(6) The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to any one of (1) to (5), wherein the nucleic acid molecule is an siRNA.
(7) A pharmaceutical composition for treating cancer, comprising the agent for suppressing the expression of an immune checkpoint factor in cancer cells according to any one of (1) to (6) and a pharmacologically acceptable additive.
また、本発明の他の態様としては、癌細胞における免疫チェックポイント因子の発現抑制剤として使用するための、（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子、又は(II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物や、（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子、又は(II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物の、免疫チェックポイント因子の発現抑制剤の調製における使用や、（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子、又は(II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物を対象に投与することを特徴とする、癌細胞における免疫チェックポイント因子の発現抑制方法を挙げることができる。 In another embodiment of the present invention, there is provided a nucleic acid molecule (I) targeting the RAB3B gene and suppressing the expression of the RAB3B gene, or (II) for use as an agent for suppressing the expression of an immune checkpoint factor in cancer cells. ) A low-molecular compound that binds to the RAB3B protein shown in SEQ ID NO: 1, (I) a nucleic acid molecule that targets the RAB3B gene and suppresses the expression of the RAB3B gene, or (II) binds to the RAB3B protein shown in SEQ ID NO: 1 The use of low molecular weight compounds in the preparation of an agent for suppressing the expression of an immune checkpoint factor, (I) a nucleic acid molecule that targets the RAB3B gene and suppresses the expression of the RAB3B gene, or (II) RAB3B represented by SEQ ID NO: 1. A method for suppressing the expression of an immune checkpoint factor in cancer cells, which comprises administering to a subject a low-molecular compound that binds to a protein.
本発明の癌細胞における免疫チェックポイント因子の発現抑制剤を用いることにより、癌細胞、特に癌幹細胞における免疫チェックポイント分子のリガンドであるPD-L1及びPD-L2の発現を抑制することが可能となる。 By using the agent for suppressing the expression of an immune checkpoint factor in cancer cells of the present invention, it is possible to suppress the expression of PD-L1 and PD-L2, which are ligands for immune checkpoint molecules in cancer cells, particularly cancer stem cells. Become.
本発明の癌細胞における免疫チェックポイント因子の発現抑制剤としては、
（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子；
（II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物；
の（I）又は（II）のいずれかを有効成分とする、癌細胞における免疫チェックポイント因子の発現抑制剤であれば特に制限されず、ここで、「RAB3B」(Entrez Gene ID:5865, mRNA: NM_002867.3, protein: NP_002858.2)は、Rasスーパーファミリーに属し、炎症反応に依存して誘導されることや、下垂体培養細胞において分泌顆粒の細胞内輸送の過程に関与していることが知られている。
As the expression inhibitor of the immune checkpoint factor in the cancer cells of the present invention,
(I) a nucleic acid molecule that targets the RAB3B gene and suppresses the expression of the RAB3B gene;
(II) a low-molecular compound that binds to the RAB3B protein represented by SEQ ID NO: 1;
The inhibitor is not particularly limited as long as it is an inhibitor of the expression of an immune checkpoint factor in cancer cells, which contains any one of (I) and (II) as an active ingredient. Here, "RAB3B" (Entrez Gene ID: 5865, mRNA : NM_002867.3, protein: NP_002858.2) belongs to the Ras superfamily and is induced depending on the inflammatory response, and is involved in the intracellular transport of secretory granules in pituitary culture cells It has been known.
また、本発明の癌治療用医薬組成物としては、上記本件免疫チェックポイント因子の発現抑制剤と、薬理学的に許容される添加物を含有する癌治療用医薬組成物であれば特に制限されない。 Further, the pharmaceutical composition for cancer treatment of the present invention is not particularly limited as long as it is a pharmaceutical composition for cancer treatment containing the above-mentioned inhibitor of the present immune checkpoint factor and a pharmacologically acceptable additive. .
上記「癌細胞」、「癌幹細胞」、又は「抗癌剤」における癌としては、固形癌でも血液癌でもよく、肝癌（肝細胞癌、又は胆管細胞癌）、膵臓癌、胃癌、食道癌、腺癌、扁平上皮癌、腺扁平上皮癌、未分化癌、大細胞癌、小細胞癌、皮膚癌、乳癌、前立腺癌、膀胱癌、膣癌、頸部癌、子宮癌、腎臓癌、脾臓癌、肺癌、気管癌、気管支癌、大腸癌（直腸癌、又は結腸癌）、小腸癌、胆嚢癌、胆道癌、精巣癌、卵巣癌等の癌や、骨組織、軟骨組織、脂肪組織、筋組織、血管組織及び造血組織の癌のほか、軟骨肉腫、ユーイング肉腫、悪性血管内皮腫、悪性シュワン腫、骨肉腫、軟部組織肉腫等の肉腫や、肝芽腫、髄芽腫、腎芽腫、神経芽腫、膵芽腫、胸膜肺芽腫、網膜芽腫等の芽腫や、胚細胞腫瘍や、リンパ腫や、白血病を挙げることができる。 The cancer in the above “cancer cell”, “cancer stem cell”, or “anticancer agent” may be a solid cancer or a hematological cancer, a liver cancer (hepatocellular carcinoma or cholangiocellular carcinoma), a pancreatic cancer, a gastric cancer, an esophageal cancer, an adenocarcinoma , Squamous cell carcinoma, glandular squamous cell carcinoma, undifferentiated cancer, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, kidney cancer, spleen cancer, lung cancer , Tracheal cancer, bronchial cancer, colorectal cancer (rectal cancer or colon cancer), small intestine cancer, gallbladder cancer, biliary tract cancer, testicular cancer, ovarian cancer, etc., bone tissue, cartilage tissue, adipose tissue, muscle tissue, blood vessels Tissue and hematopoietic tissue cancer, as well as sarcomas such as chondrosarcoma, Ewing sarcoma, malignant hemangioendothelioma, malignant schwanoma, osteosarcoma, soft tissue sarcoma, hepatoblastoma, medulloblastoma, nephroblastoma, and neuroblastoma , Blastomas such as pancreatic blastoma, pleural pulmonary blastoma, and retinoblastoma, germ cell tumors, lymphomas, and leukemias.
上記「癌細胞における免疫チェックポイント因子」としては、癌細胞で発現し、免疫監視機構に関与する受容体のリガンドを意味する。具体的にはPD-L1又はPD-L2等のT細胞の免疫寛容を誘導するPD-1のリガンドや、TIM-3のリガンドであるCEACAM1、NKG2DのリガンドであるRAE1を挙げることができ、PD-L1又はPD-L2を好適に挙げることができる。なお、上記癌細胞における免疫チェックポイント因子の発現が抑制されることで、免疫寛容が解除され、その結果、免疫細胞により癌細胞が破壊される。 The “immune checkpoint factor in cancer cells” means a ligand of a receptor that is expressed in cancer cells and is involved in an immune surveillance mechanism. Specifically, PD-L1 or PD-L2, such as PD-1 ligand for inducing immune tolerance of T cells, TIM-3 ligand CEACAM1, NKG2D ligand RAE1, and the like. -L1 or PD-L2 can be suitably mentioned. In addition, suppression of the expression of the immune checkpoint factor in the above cancer cells releases immune tolerance, and as a result, the cancer cells are destroyed by the immune cells.
上記「RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子」としてはRAB3B遺伝子の全長又は部分配列と結合し、RAB3B遺伝子の発現を抑制できる核酸分子であるかぎり特に制限されず、RAB3B遺伝子の転写を抑制する核酸分子や、RAB3B遺伝子を破壊する核酸分子や、RAB3B遺伝子の翻訳を抑制する核酸分子を挙げることができる。具体的にはRAB3B遺伝子の全長又は部分配列を標的とするsiRNA、microRNA、又はshRNAや、RAB3B遺伝子の全長又は部分配列を標的とするguide RNAや、RAB3B遺伝子の全長又は部分配列を標的とするアンチセンス核酸や、RAB3B遺伝子の全長又は部分配列を標的とするリボザイム等を挙げることができる。上記「siRNA」としては、例えば配列番号2に示されるセンス配列と配列番号3に示されるアンチセンス配列とから形成されるsiRNA、又は配列番号4に示されるセンス配列と配列番号5に示されるアンチセンス配列とから形成されるsiRNAを挙げることができる。また、Cas9タンパク質をゲノムに標的化させるguide RNA配列としては、例えば配列番号10に示されるguide RNA配列を挙げることができる。guide RNA配列を用いる場合には、Cas9又はその改変物(エンドヌクレアーゼ活性から転写調節活性への置き換え等) のmRNAと共に用いることが好ましい。さらに、microRNAとしては、例えば配列番号6に示されるhas-miR-15a-5p(microRNA database（http://www.mirbase.org/）ID:MIMAT0000068)や、has-miR-15a-5pのミミック RNAを挙げることができる。また、上記siRNA、上記guide RNA配列、又は上記microRNAの塩基配列と少なくとも85%以上、好ましくは90%以上、より好ましくは95%以上、さらに好ましくは98%以上の同一性を示す塩基配列からなり、かつRAB3B遺伝子を標的としてRAB3B遺伝子の発現を抑制できるRNAを挙げることもできる。なお、配列番号2における3’末端のtt(tはチミン)、配列番号3における3’末端のcc(cはシトシン)、配列番号4における3’末端のtt、配列番号5における3’末端のgg(gはグアニン)はRNAをヌクレアーゼから保護するためのオーバーハング配列である。また、上記RAB3B遺伝子には、RAB3Bタンパク質の構造遺伝子だけでなく、その構造遺伝子の5’-UTR及び3’-UTRやプロモーター領域、エンハンサー領域などの転写調節領域も含む。 The `` targeted RAB3B gene, the nucleic acid molecule which suppresses the expression of the RAB3B gene '' is not particularly limited as long as it is a nucleic acid molecule capable of binding to the full length or partial sequence of the RAB3B gene and suppressing the expression of the RAB3B gene. , A nucleic acid molecule that disrupts the RAB3B gene, and a nucleic acid molecule that suppresses the translation of the RAB3B gene. Specifically, siRNA, microRNA, or shRNA targeting the full-length or partial sequence of the RAB3B gene, guide RNA targeting the full-length or partial sequence of the RAB3B gene, and anti-RNA targeting the full-length or partial sequence of the RAB3B gene Examples include a sense nucleic acid and a ribozyme targeting the full length or partial sequence of the RAB3B gene. As the “siRNA”, for example, an siRNA formed from the sense sequence shown in SEQ ID NO: 2 and the antisense sequence shown in SEQ ID NO: 3, or the sense sequence shown in SEQ ID NO: 4 and the antisense shown in SEQ ID NO: 5 And an siRNA formed from the sense sequence. The guide RNA sequence for targeting the Cas9 protein to the genome includes, for example, the guide RNA sequence shown in SEQ ID NO: 10. When the guide RNA sequence is used, it is preferably used together with mRNA of Cas9 or a modified product thereof (eg, replacement of endonuclease activity with transcription regulatory activity). Further, as microRNA, for example, has-miR-15a-5p shown in SEQ ID NO: 6 (microRNA database (http://www.mirbase.org/) ID: MIMAT0000068) or mimic of has-miR-15a-5p RNA can be mentioned. Further, the siRNA, the guide RNA sequence, or a base sequence of at least 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more identity with the base sequence of the microRNA. And RNA capable of suppressing the expression of the RAB3B gene by targeting the RAB3B gene. In addition, tt at the 3 ′ end in SEQ ID NO: 2 (t is thymine), cc at the 3 ′ end in SEQ ID NO: 3 (c is cytosine), tt at the 3 ′ end in SEQ ID NO: 4, 3 ′ end in SEQ ID NO: 5 gg (g is guanine) is an overhang sequence for protecting RNA from nucleases. Further, the RAB3B gene includes not only the structural gene of the RAB3B protein but also a transcription regulatory region such as a 5'-UTR and a 3'-UTR of the structural gene, a promoter region and an enhancer region.
上記「RAB3Bタンパク質に結合する低分子化合物」としては、RAB3Bタンパク質の少なくとも一部に結合する低分子の化合物であればよく、ゲニステイン(Genistein)等のチロシンキナーゼ(tyrosine kinase)阻害剤を挙げることができ、かかる低分子化合物は、公知の方法によりスクリーニングして得ることができ、天然の化合物であっても又は人工の化合物のいずれであっても良い。上記スクリーニング方法としては、例えば、RAB3Bタンパク質又はその部分ペプチドと被検低分子化合物を接触させて、RAB3Bタンパク質又はその部分ペプチドと被検化合物との結合活性を調べ、RAB3Bタンパク質又はその部分ペプチドと結合する化合物を選択する方法を挙げることができる。 The `` low-molecular compound that binds to the RAB3B protein '' may be a low-molecular compound that binds to at least a part of the RAB3B protein, and may include a tyrosine kinase (tyrosine kinase) inhibitor such as genistein (Genistein). Such a low-molecular compound can be obtained by screening by a known method, and may be a natural compound or an artificial compound. As the above screening method, for example, by contacting the test low molecular compound with the RAB3B protein or its partial peptide, to examine the binding activity between the RAB3B protein or its partial peptide and the test compound, and binding to the RAB3B protein or its partial peptide And a method of selecting a compound to be used.
上記「薬理的に許容される添加剤」としては、生理食塩水、緩衝生理食塩水、細胞培養培地、デキストロース、注射用水、グリセロール、エタノール及びこれらの組合せ、安定剤、可溶化剤及び界面活性剤、緩衝剤及び防腐剤、等張化剤、充填剤、並びに潤滑剤を挙げることができる。 Examples of the “pharmacologically acceptable additive” include physiological saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants , Buffers and preservatives, tonicity agents, fillers, and lubricants.
上記癌治療用医薬組成物には抗癌剤を含有してもよく、かかる抗癌剤としては、ドキソルビシン、イダルビジン、マイトマイシンＣ等の抗腫瘍性抗生物質、ドセタキセル、ビンクリスチン等の微小管阻害剤、カルボプラチン、シスプラチン、オキサリプラチン等のプラチナ製剤、ボリノスタット等のヒストン脱アセチル化酵素（HDAC）阻害剤、スニチニブ、イマチニブ、ゲフェチニブ、エルロチニブ、アファチニブ、ダサチニブ、トラメチニブ等のキナーゼ阻害剤、イリノテカン、エトポシド等のトポイソメラーゼ阻害剤、シクロスポリン、タクロリムス等のカルシニューリン阻害薬、シクロホスファミド、ベンダムスチン、イオスファミド、ダカルバジン等のアルキル化薬、ペントスタチン、フルダラビン、クラドリビン、メソトレキセート、５−フルオロウラシル、６−メルカプトプリン、エノシタビン等の代謝拮抗薬、リツキシマブ、セツキシマブ、トラスツズマブ等の分子標的薬、ボルテゾミブ等のプロテアソーム阻害剤、タモキシフェン、ビカルダミド等のホルモン療法薬、ニボルマブ、ペンブロリズマブ、ピジリズマブ等の抗ＰＤ−１抗体、アテゾリズマブ、アベルマブ、又はデュルバルマブ、ＢＭＳ−９３６５５９等の抗ＰＤ−Ｌ１抗体、イピリムマブ又はトレメリムマブ等の抗ＣＴＬＡ−４抗体を挙げることができ、抗腫瘍性抗生物質、微小管阻害剤、プラチナ製剤、ヒストン脱アセチル化酵素（HDAC）阻害剤、キナーゼ阻害剤、トポイソメラーゼ阻害剤、抗ＰＤ−１抗体、抗ＣＴＬＡ−４抗体を好適に挙げることができる。 The pharmaceutical composition for treating cancer may contain an anticancer agent, such anticancer agents such as doxorubicin, idarubidin, antitumor antibiotics such as mitomycin C, docetaxel, microtubule inhibitors such as vincristine, carboplatin, cisplatin, Platinum preparations such as oxaliplatin, histone deacetylase (HDAC) inhibitors such as vorinostat, sunitinib, imatinib, gefetinib, erlotinib, kinase inhibitors such as afatinib, dasatinib, trametinib, topoisomerin inhibitors such as irinotecan and etoposide , Tacrolimus and other calcineurin inhibitors, cyclophosphamide, bendamustine, iosfamide, alkylating drugs such as dacarbazine, pentostatin, fludarabine, cladribine, methotrexate , 5-fluorouracil, 6-mercaptopurine, anocitabine, etc., molecular target drugs such as rituximab, cetuximab, trastuzumab, proteasome inhibitors such as bortezomib, hormone therapy drugs such as tamoxifen, bicardamide, nivolumab, pembrolizumab, pizilizumab etc. Anti-PD-1 antibody, atezolizumab, averumab, or anti-PD-L1 antibody such as durvalumab, BMS-936559, anti-CTLA-4 antibody such as ipilimumab or tremelimumab, antitumor antibiotics, microtubule inhibition Agents, platinum preparations, histone deacetylase (HDAC) inhibitors, kinase inhibitors, topoisomerase inhibitors, anti-PD-1 antibodies, and anti-CTLA-4 antibodies.
上記癌治療用医薬組成物は、当業者に既知の方法を用いて、癌の治療を必要とする被検体に投与することができ、投与方法としては、静脈内、腫瘍内、皮内、皮下、筋肉内、腹腔内、動脈内、髄内、心臓内、関節内、滑液嚢内、頭蓋内、髄腔内、及びくも膜下（髄液）への注射を挙げることができる。 The pharmaceutical composition for cancer treatment can be administered to a subject in need of treatment for cancer using a method known to those skilled in the art. Examples of the administration method include intravenous, intratumoral, intradermal, and subcutaneous. Intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and subarachnoid (cerebrospinal fluid) injections.
上記癌細胞における免疫チェックポイント因子の発現抑制剤又は上記癌治療用医薬組成物の投与量は、癌の種類、位置、重症度、治療を受ける被検体の年齢、体重及び状態等に応じて適宜調整できるが、好ましくは、１回の投与において体重1kgあたりRAB3B遺伝子の発現を抑制する核酸分子として0.01μg-1000μg、より好ましくは0.1μg-100μgを挙げることができ、配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物として0.01μg-100μg、好ましくは0.1μg-10μgを挙げることができる。 The dose of the agent for suppressing the expression of the immune checkpoint factor in the cancer cells or the pharmaceutical composition for treating cancer is appropriately determined depending on the type, location, severity of the cancer, age, weight, and condition of the subject to be treated. Although it can be adjusted, preferably, a nucleic acid molecule that suppresses the expression of the RAB3B gene per 1 kg of body weight in a single administration is 0.01 μg-1000 μg, more preferably 0.1 μg-100 μg, and RAB3B represented by SEQ ID NO: 1. Examples of the low molecular compound that binds to a protein include 0.01 μg to 100 μg, preferably 0.1 μg to 10 μg.
投与する上記癌細胞における免疫チェックポイント因子の発現抑制剤又は上記癌治療用医薬組成物は、1日4回、3回、2回又は1回、1日おき、2日おき、3日おき、4日おき、5日おき、週1回、7日おき、8日おき、9日おき、週2回、月1回又は月2回独立して投与する方法を挙げることができる。 The inhibitor of the expression of the immune checkpoint factor in the cancer cells to be administered or the pharmaceutical composition for treating cancer is four times a day, three times, twice or once, every other day, every two days, every three days, Examples of the method include independent administration every 4 days, every 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, once a month or twice a month.
上記癌細胞における免疫チェックポイント因子の発現抑制剤又は癌治療用医薬組成物は、他の抗癌剤と併用して用いることこができる。前記「他の抗癌剤と併用して用いる」方法としては、該他の抗癌剤を用いて処理し、その後上記癌細胞における免疫チェックポイント因子の発現抑制剤又は癌治療用医薬組成物を用いる方法や、上記癌細胞における免疫チェックポイント因子の発現抑制剤又は癌治療用医薬組成物と該他の抗癌剤を同時に用いる方法や、上記癌細胞における免疫チェックポイント因子の発現抑制剤又は癌治療用医薬組成物を用いて処理し、その後該他の抗癌剤を用いる方法を挙げることができる。また、上記癌細胞における免疫チェックポイント因子の発現抑制剤又は癌治療用医薬組成物と該他の抗癌剤と併用した場合には、薬物の治療効果がより向上すると共に、該薬物の投与回数又は投与量を減らすことで、該薬物による副作用を低減させることが可能となる。 The agent for suppressing the expression of an immune checkpoint factor in cancer cells or the pharmaceutical composition for treating cancer can be used in combination with other anticancer agents. As the method of `` using in combination with another anticancer agent '', a method of treating with the other anticancer agent and then using a pharmaceutical composition for suppressing the expression of an immune checkpoint factor in the cancer cells or a cancer treatment, A method for simultaneously using the agent for suppressing the expression of an immune checkpoint factor in cancer cells or a pharmaceutical composition for treating cancer and the other anticancer agent, or the method for suppressing the expression of an immune checkpoint factor in cancer cells or the pharmaceutical composition for treating cancer. And then using the other anticancer agent. In addition, when used together with the other anticancer drug and an inhibitor of the expression of an immune checkpoint factor in the above cancer cells or a pharmaceutical composition for treating cancer, the therapeutic effect of the drug is further improved, and the number of administrations or administration of the drug is increased. By reducing the amount, side effects due to the drug can be reduced.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの
例示に限定されるものではない。
Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these exemplifications.
［実施例１］
（SK-HEP-1細胞株からの浮遊細胞塊の誘導）
本発明者らが以前開示した癌幹細胞誘導法(上記特許文献1)を用い、肝癌由来の肝癌細胞株SK-HEP-1より、浮遊細胞塊（sphere）形成能を有する癌幹細胞を誘導した。具体的には以下に示すとおりである。
[Example 1]
(Induction of suspended cell mass from SK-HEP-1 cell line)
Using the cancer stem cell derivation method previously disclosed by the present inventors (Patent Document 1), a cancer stem cell having a floating cell mass (sphere) forming ability was induced from a liver cancer cell line SK-HEP-1 derived from liver cancer. Specifically, it is as shown below.
まず、低分化型肝細胞癌由来の肝癌細胞株であるSK-HEP-1細胞株の生細胞数をトリパンブルー染色より計測し、1.0×10５個／mlとなるように以下に示すNSF（neural survival factor-1）含有無血清培地（neural stem cell培地：以下、「NSC培地」ともいう）に懸濁した後、ベントキャップタイプフラスコ（BDファルコン社製）又は超低接着表面フラスコ カントネック ベントキャップ（Corning社製）に播種し、37℃、5%CO2条件下で培養した。培養後7日目には、SK-HEP-1細胞株由来の癌幹細胞様細胞塊のうち浮遊した細胞塊の細胞（以下、「SK-sphere」ともいう）を誘導した。NSC培地は、以下の成分A、B、及びCからなるものを作製して用いた。 First, the viable cell count of the SK-HEP-1 cell line, which is a hepatoma cell line derived from poorly differentiated hepatocellular carcinoma, was measured by trypan blue staining, and NSF (NSF) shown below was adjusted to 1.0 × 10 5 cells / ml. Neural stem cell medium (neural stem cell medium: hereinafter, also referred to as "NSC medium") is suspended in a vent cap type flask (manufactured by BD Falcon) or an ultra-low adhesion surface flask. The cells were seeded in caps (manufactured by Corning) and cultured under conditions of 37 ° C. and 5% CO 2 . On the 7th day after the culture, cells of a suspended cell mass (hereinafter, also referred to as “SK-sphere”) among the cancer stem cell-like cell mass derived from the SK-HEP-1 cell line were induced. The NSC medium prepared and used was composed of the following components A, B, and C.
（１）成分A
DMEM/F12（シグマ−アルドリッチ社製） 86mL
1M Hepes（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 900μL
Antibiotic/antimycotic liquid(100倍濃度) 900μL
30% グルコース 1.7mL
（２）成分B
DMEM/F12培地（シグマ−アルドリッチ社製） 8.6mL
30%グルコース（シグマ−アルドリッチ社製） 200μL
トランスフェリン（シグマ−アルドリッチ社製）10mg+H2O 200μL
インスリン（シグマ−アルドリッチ社製） 2.5mg+0.1N HCl 100μL(先にインスリンを溶解) +H2O 900μL(溶解後に加える) 計1mL
プトレシン（Alexis Biochemicals社製） 19.33mg
0.3mM 亜セレン酸ナトリウム（シグマ−アルドリッチ社製） 10μL
2mM プロゲステロン（シグマ−アルドリッチ社製） 1μL
（３）成分C
200μg/mL ヒトEGF（シグマ−アルドリッチ社製） 10μL
4μg/mL Basic FGF（和光純薬工業社製） 500μL
1mg/mL ヘパリン（シグマ−アルドリッチ社製） 200μL
10μg/mL LIF（ケミコン社製） 100μL
NSF-1（50倍濃度）（カンブレックス社製） 2mL（最終濃度；2%［w/v］）
60mg/mL Ｎ−アセチルシステイン（N-acetylcysteine）（シグマ−アルドリッチ社製） 100μL
(1) Component A
DMEM / F12 (Sigma-Aldrich) 86mL
1M Hepes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) 900μL
Antibiotic / antimycotic liquid (100 times concentration) 900μL
1.7% 30% glucose
(2) Component B
DMEM / F12 medium (Sigma-Aldrich) 8.6mL
30% glucose (Sigma-Aldrich) 200μL
Transferrin (Sigma - Aldrich) 10mg + H 2 O 200μL
Insulin (Sigma-Aldrich) 2.5mg + 0.1N HCl 100µL (dissolve insulin first) + H 2 O 900µL (added after dissolution) Total 1mL
Putrescine (Alexis Biochemicals) 19.33mg
0.3 mM Sodium selenite (Sigma-Aldrich) 10 μL
2 μM progesterone (Sigma-Aldrich) 1 μL
(3) Component C
200μg / mL Human EGF (Sigma-Aldrich) 10μL
4μg / mL Basic FGF (Wako Pure Chemical Industries) 500μL
1mg / mL Heparin (Sigma-Aldrich) 200μL
10μg / mL LIF (Chemicon) 100μL
NSF-1 (50 times concentration) (Cambrex) 2mL (final concentration; 2% [w / v])
60 mg / mL N-acetylcysteine (Sigma-Aldrich) 100 μL
なお、ここでは約100mLの無血清培地を作製する場合の組成を記載している。また、無血清培地は、成分A、B及びCをそれぞれ個別に作製した後、混合して作製した。 Here, the composition for preparing about 100 mL of a serum-free medium is described. The serum-free medium was prepared by separately preparing components A, B, and C and then mixing them.
上記で誘導したSK-sphereを免疫不全マウスNOD-Rag1null IL2r null double mutant mice (NRG mice)の脾臓に27Gの注射針を用いて注入した後に脾臓を摘出し、8週間後にマウスを開腹し肝腫瘍形成を確認することで、上記SK-sphereは血行性肝転移能を有することを確認した。 The SK-sphere induced above was injected into the spleen of the immunodeficient mouse NOD-Rag1 null IL2r null double mutant mice (NRG mice) using a 27G injection needle, and the spleen was removed. By confirming tumor formation, it was confirmed that the SK-sphere had hematogenous liver metastasis ability.
（SK-HEP-1細胞株の培養）
SK-HEP-1細胞株を1.0×10５個/mLとなるように10%ウシ胎児血清（FBS:fetal bovine serum）を含むDMEM培地（以下、「DMEM培地」という）に懸濁した後、培養シャーレに播種し、37℃、5% CO2条件下で3日間培養した。DMEM培地で培養したSK-HEP-1細胞株（以下、「SK-HEP-1」ともいう）は培養シャーレに接着する接着性の癌細胞株である。
(Culture of SK-HEP-1 cell line)
After suspending the SK-HEP-1 cell line in a DMEM medium containing 10% fetal bovine serum (FBS) at a concentration of 1.0 × 10 5 cells / mL (hereinafter referred to as “DMEM medium”), The cells were seeded in a culture dish and cultured at 37 ° C. under 5% CO 2 for 3 days. The SK-HEP-1 cell line cultured in a DMEM medium (hereinafter, also referred to as “SK-HEP-1”) is an adhesive cancer cell line that adheres to a culture dish.
（HuH7細胞株の培養）
高分化型肝細胞癌由来の細胞株であるHuH7細胞株を、上記NSC培地、又は上記DMEM培地で培養した。具体的には、HuH7細胞株を1.0×105個/mLとなるように上記NSC培地又は上記DMEM培地に懸濁した後、培養シャーレに播種し、37℃、5%CO2条件下でそれぞれ7日間又は3日間培養した。上記NSC培地及び上記DMEM培地で培養したHuH7細胞株（それぞれ、以下「HuH7/NSC」、「HuH7/DMEM」ともいう）はいずれも培養シャーレに接着し、浮遊細胞塊は形成されなかった。したがって、NSC培地を用いたとしても、低分化型肝癌由来のSK-HEP-1細胞株を培養した場合には浮遊細胞塊を形成するのに対し、高分化型肝癌由来のHuH7細胞株を培養した場合には浮遊細胞塊を形成しないことが明らかとなった。
［実施例２］
（転移性肝内再発に特異的な遺伝子の同定）
実施例1で得られたSK-sphere、SK-HEP-1、HuH7/NSC、HuH7/DMEMそれぞれの肝細胞癌由来細胞、及び摘除手術後5年以上肝内再発無しHCC患者（HCC without IHR(≧5year)）若しくは摘除手術後1年以内転移性肝内再発ありHCC患者（HCC with IHR(≦1year)）からの摘除標本における癌部組織及び周辺非癌部組織のmRNA発現を解析することで、原発巣除去後の転移性肝内再発に特異的な遺伝子の同定を行った。
(Culture of HuH7 cell line)
A HuH7 cell line, which is a cell line derived from a well-differentiated hepatocellular carcinoma, was cultured in the NSC medium or the DMEM medium. Specifically, after suspending the HuH7 cell line in the NSC medium or the DMEM medium so as to be 1.0 × 10 5 cells / mL, the cells were seeded in a culture dish, and each was cultured at 37 ° C. under 5% CO 2 conditions. Culture was performed for 7 days or 3 days. Both HuH7 cell lines cultured in the NSC medium and the DMEM medium (hereinafter also referred to as “HuH7 / NSC” and “HuH7 / DMEM,” respectively) adhered to the culture dish, and no floating cell mass was formed. Therefore, even when the NSC medium is used, when the SK-HEP-1 cell line derived from poorly differentiated liver cancer is cultured, a floating cell mass is formed, whereas the HuH7 cell line derived from highly differentiated liver cancer is cultured. In this case, it was found that no floating cell mass was formed.
[Example 2]
(Identification of genes specific to metastatic intrahepatic recurrence)
SK-sphere, SK-HEP-1, HuH7 / NSC, and HuH7 / DMEM hepatocellular carcinoma-derived cells obtained in Example 1, and HCC patients without intrahepatic recurrence for 5 years or more after excision surgery (HCC without IHR ( ≧ 5year)) or by analyzing mRNA expression in cancerous tissues and surrounding non-cancerous tissues in resected specimens from HCC patients with metastatic intrahepatic recurrence within 1 year after resection surgery (HCC with IHR (≦ 1year)). Then, a gene specific for metastatic intrahepatic recurrence after removal of the primary tumor was identified.
（mRNAの抽出）
miRNeasy（QIAGEN社製）を用いて、実施例１で作製したSK-sphere、SK-HEP-1、HuH7/NSC、HuH7/DMEMそれぞれの癌由来細胞株、及び摘除手術後5年以上肝内再発無しHCC患者（HCC without IHR(≧5year)）及び摘除手術後1年以内転移性肝内再発有りHCC患者（HCC with IHR(≦1year)）からの摘除原発巣標本（それぞれn=5）における癌部組織からtotal RNAを抽出した。
(Extraction of mRNA)
Using miRNeasy (manufactured by QIAGEN), cancer-derived cell lines of SK-sphere, SK-HEP-1, HuH7 / NSC, and HuH7 / DMEM prepared in Example 1, and intrahepatic recurrence for more than 5 years after removal surgery Cancer in the resected primary specimens (n = 5 each) from patients without HCC without HCC (HCC without IHR (≧ 5year)) and those with metastatic intrahepatic recurrence within 1 year after resection surgery (HCC with IHR (≦ 1year)) Total RNA was extracted from partial tissues.
（mRNAの発現解析）
TruSeq Stranded Total RNA with Ribo-Zero Gold LT Sample Prep kit（イルミナ社製）及びNextSeq500（イルミナ社製）を用いて、そのプロトコルに従ってribosomal RNA除去後にRNAシークエンスを行い、26,475遺伝子の網羅的なmRNA発現解析を行った。NextSeq 500より得られたfastqファイルからcutadaptによるデータトリミング、FastQCによるクオリティ確認を経てSTARによるhg38ヒトリファレンスゲノムへのマッピングを行った。26,475遺伝子それぞれにおけるリードカウント数について、R softwareにおけるTCCパッケージを用いて正規化及び群間比較を行った。群間比較に先立って正規化後に何れの分においても発現量が低い(read count=10)遺伝子は除外した。発現変動遺伝子(Differentially expressed Genes; DEGs)の検出にはGLM LRT法を用いた。解析にあたっては、(a)SK-sphereとSK-HEP-1、(b)SK-sphereとHuH7/NSC、(c)HCC with IHR(≦1year)vs HCC without IHR(≧5year)におけるmRNAの発現量を比較して、転移性肝内再発有り群及びSK-sphereに共通して発現亢進を示す遺伝子候補を選択した。選択基準は（Fold-change）2.0、q値〈0.05）とした。q値は、FDR多重検定（multiple-testing correction）により算出した。
(MRNA expression analysis)
Using TruSeq Stranded Total RNA with Ribo-Zero Gold LT Sample Prep kit (Illumina) and NextSeq500 (Illumina), perform RNA sequencing after removing ribosomal RNA according to the protocol, and perform comprehensive mRNA expression analysis of 26,475 genes Was done. From the fastq file obtained from NextSeq 500, data was trimmed by cutadapt, quality was confirmed by FastQC, and mapping to the hg38 human reference genome was performed by STAR. The read counts of each of the 26,475 genes were normalized and compared between groups using the TCC package in R software. Genes with low expression levels (read count = 10) in any of the fractions after normalization prior to intergroup comparison were excluded. The GLM LRT method was used to detect differentially expressed genes (DEGs). In the analysis, mRNA expression in (a) SK-sphere and SK-HEP-1, (b) SK-sphere and HuH7 / NSC, (c) HCC with IHR (≦ 1year) vs HCC without IHR (≧ 5year) By comparing the amounts, gene candidates showing increased expression in common to the group with metastatic intrahepatic recurrence and the SK-sphere were selected. The selection criteria were (Fold-change) 2.0, q value <0.05. The q value was calculated by FDR multiple-testing correction.
（MA plot解析）
さらに、上記それぞれの発現量解析の結果を統合してMA plot解析(SK-sphere vs SK-HEP-1及びShort-DFS(Disease-free survival)vs Long-DFS in Tumor)を行い、SK-sphere及び転移性肝内再発有り群に共通して発現亢進を示す遺伝子の探索を行った。肝癌におけるDFSは無再発生存期間を意味し、RFS (relapse-free survival)と同義である。上記遺伝子の探索手法の概略を図1に、MA plot解析の結果を図2及び3に示す。図2及び3中、横軸はSK-sphereとSK-HEP-1での総カウント数(mRNAの発現量に相当)又はShort-DFSとLong-DFSでの総カウント数のLog2値、縦軸はSK-sphere/SK-HEP-1又はShort-DFS/Long-DFS発現比のLog2値を表す。
(MA plot analysis)
Furthermore, the results of the respective expression level analyzes are integrated and MA plot analysis (SK-sphere vs SK-HEP-1 and Short-DFS (Disease-free survival) vs Long-DFS in Tumor) is performed, and SK-sphere Further, a gene showing an increased expression in common with the group having metastatic intrahepatic recurrence was searched. DFS in liver cancer means relapse-free survival and is synonymous with RFS (relapse-free survival). FIG. 1 shows an outline of the above gene search method, and FIGS. 2 and 3 show the results of MA plot analysis. 2 and 3, the horizontal axis is the total count number in SK-sphere and SK-HEP-1 (corresponding to the expression level of mRNA) or the Log2 value of the total count number in Short-DFS and Long-DFS, vertical axis Represents the Log2 value of the expression ratio of SK-sphere / SK-HEP-1 or Short-DFS / Long-DFS.
図3に示すMA plot解析により、転移性肝内再発有り群及びSK-sphereに共通して発現亢進を示す遺伝子、すなわち、原発巣除去後の術後転移性肝内再発に特異的な遺伝子の候補として図3の矢印のスポットに位置するsmall GTPaseをコードする遺伝子RAB3Bを同定した。 According to the MA plot analysis shown in FIG. 3, the gene showing expression enhancement commonly in the group with metastatic intrahepatic recurrence and SK-sphere, that is, the gene specific to postoperative metastatic intrahepatic recurrence after removal of the primary tumor As a candidate, the gene RAB3B encoding small GTPase located at the spot indicated by the arrow in FIG. 3 was identified.
［実施例３］
（mRNA発現レベルの比較）
１．RNAシークエンス
実施例２におけるmRNAの発現解析によるSK-sphere、SK-HEP-1、HuH7/NSC、HuH7/DMEMそれぞれの癌由来細胞株からのRAB3B ｍRNA発現をまとめたグラフを図4A(a)に示す。図4A(a)に示すように、SK-sphereはSK-HEP-1と比較して2.3倍もRAB3BのmRNAが発現していることが明らかとなった。一方、HuH7においては、培養条件にかかわらずRAB3BのmRNA発現レベルはSK-HEP-1におけるRAB3BのmRNA発現レベルよりも低かった。
[Example 3]
(Comparison of mRNA expression levels)
1. RNA sequencing FIG. 4A (a) is a graph summarizing RAB3B mRNA expression from cancer-derived cell lines of SK-sphere, SK-HEP-1, HuH7 / NSC, and HuH7 / DMEM by mRNA expression analysis in Example 2. Show. As shown in FIG. 4A (a), it was revealed that SK-sphere expressed RAB3B mRNA 2.3 times as much as SK-HEP-1. On the other hand, in HuH7, RAB3B mRNA expression level was lower than RAB3B mRNA expression level in SK-HEP-1 regardless of culture conditions.
２．定量PCR
定量PCRによりSK-sphere、SK-HEP-1、HuH7/NSC、HuH7/DMEMそれぞれの癌由来細胞株、及び摘除手術後2年を超えて肝内再発無しHCC患者（HCC without IHR(＞2year)）若しくは摘除手術後1年以内転移性肝内再発有りHCC患者（HCC with IHR(≦1year)）からの摘除標本（それぞれn=5）における癌部組織からのRAB3B mRNA発現量を解析した。定量PCR解析は、以下の方法で行った。
2. QPCR
Quantitative PCR by SK-sphere, SK-HEP-1, HuH7 / NSC, HuH7 / DMEM cancer-derived cell lines, and HCC patients with no intrahepatic recurrence more than 2 years after resection surgery (HCC without IHR (> 2year) ) Or RAB3B mRNA expression level from cancerous tissue in resected specimens (n = 5 each) from HCC patients with metastatic intrahepatic recurrence within 1 year after resection surgery (HCC with IHR (≦ 1 year)). The quantitative PCR analysis was performed by the following method.
まず、miRNeasy（QIAGEN社製）を用いてtotalRNAを抽出及び精製し、さらにPrimeScript T reagent Kit with gDNA Eraser（TaKaRa Bio社製）を用いてゲノムDNA除去後にcDNAを合成した。合成したcDNAを鋳型として、LightCycler 480 Probe Master（Roche Diagnostics社製）、Universal ProbeLibrary（Roche Diagnostics社製）、及びLightCycler480 System II（Roche Diagnostics社製）を用いて定量PCRを行った。その後の統計学的解析は、R softwareを用いて行った。P値は、漸近計算した対応のないｔ−検定（unpaired t-test）により算出した。結果を図4A(b)、(c)、図4Bに示す。図4A(c)は図4(b)のSK-HEP-1、SK-sphereのウエスタンブロットの結果である。RAB3Bに対する抗体にはAnti-RAB3B Mouse monoclonalantibody (ab55655, abcam社製)を用いた。 First, total RNA was extracted and purified using miRNeasy (manufactured by QIAGEN), and cDNA was synthesized after removing genomic DNA using PrimeScript T reagent Kit with gDNA Eraser (manufactured by TaKaRa Bio). Using the synthesized cDNA as a template, quantitative PCR was performed using LightCycler 480 Probe Master (Roche Diagnostics), Universal ProbeLibrary (Roche Diagnostics), and LightCycler480 System II (Roche Diagnostics). Subsequent statistical analysis was performed using R software. P values were calculated by an unpaired t-test, which was asymptotically calculated. The results are shown in FIGS. 4A (b), (c), and FIG. 4B. FIG. 4A (c) shows the results of Western blot of SK-HEP-1 and SK-sphere in FIG. 4 (b). Anti-RAB3B Mouse monoclonal antibody (ab55655, manufactured by abcam) was used as an antibody against RAB3B.
図4A(b)、図4Bに示すように、RAB3Bは親株と比較してSK-sphereにてmRNA発現が有意に2.9倍上昇し、かつ臨床検体において5年以上肝内再発無しと比較して術後1年以内転移性肝内再発有りにおいてmRNA発現が有意に9.5倍上昇していた。さらに非癌部と比較して癌部でのmRNA発現が3.7倍高かった。HuH7においても通常培養下と比較して浮遊細胞塊誘導条件下での発現亢進を認めるが、その発現レベルはSK-HEP-1よりも低いことが明らかとなった。また、図4A(c)に示すように、タンパク質レベルでRAB3Bがsphereで発現が亢進していることが確認された。図4A(b)、(c)、図4Bの結果より、RAB3Bの発現が浮遊細胞塊形成や転移性肝内再発に関与することが明らかとなった。 As shown in FIG.4A (b) and FIG.4B, RAB3B has a significantly increased 2.9-fold mRNA expression in the SK-sphere compared to the parent strain, and in clinical samples compared to no liver recurrence for 5 years or more. MRNA expression was significantly increased by 9.5-fold within 1 year after operation with metastatic intrahepatic recurrence. Furthermore, the mRNA expression in the cancerous part was 3.7 times higher than that in the non-cancerous part. Although expression of HuH7 was enhanced under the conditions for inducing floating cell mass as compared with that in normal culture, it was revealed that the expression level was lower than that of SK-HEP-1. Further, as shown in FIG. 4A (c), it was confirmed that the expression of RAB3B was enhanced in the sphere at the protein level. The results of FIGS. 4A (b), (c), and FIG. 4B revealed that RAB3B expression is involved in the formation of floating cell mass and metastatic intrahepatic recurrence.
［実施例４］
（siRNAによるノックダウン解析）
SK-HEP-1細胞に対して、Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific社製)を用いてreverse transfection法により以下のsiRNAsをトランスフェクションした。siRNAとして、配列番号2(Sense 5’ to 3’: GCUUCAUUCUGAUGUAUGAtt)に示されるセンス配列と配列番号3（Antisense 5’ to 3’: UCAUACAUCAGAAUGAAGCcc)に示されるアンチセンス配列とから形成されるsiRAB3B#1、又は、配列番号4(Sense 5’ to 3’: GCUAUGCUGAUGACACGUUtt))に示されるセンス配列と配列番号5（Antisense 5’ to 3’: AACGUGUCAUCAGCAUAGCgg)に示されるアンチセンス配列とから形成されるsiRAB3B#2の塩基配列、及びコントロールsiRNA (siNegaCont: Silencer（登録商標） Select Negative Control No.1 siRNA; Thermo Fisher Scientific社製)を用いた。Reverse transfection法には以下の条件を適用した。1x105 cells/ 5nM each siRNA/1 μL Lipofectamine RNAiMAX Transfection Reagent/ 1mL Opti-MEM I Reduced Serum培地 (Thermo Fisher Scientific社製)。結果を図5(a)、(b)、図6、図7に示す。
[Example 4]
(Knockdown analysis by siRNA)
SK-HEP-1 cells were transfected with the following siRNAs by reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (manufactured by Thermo Fisher Scientific). As siRNA, siRAB3B # 1, formed from the sense sequence shown in SEQ ID NO: 2 (Sense 5 'to 3': GCUUCAUUCUGAUGUAUGAGAtt) and the antisense sequence shown in SEQ ID NO: 3 (Antisense 5 'to 3': UCAUACAUCAGAAUGAAGCcc), Or, SEQ ID NO: 4 (Sense 5 'to 3': GCUAUGCUGAUGACACGUUtt)) and the sense sequence shown in SEQ ID NO: 5 (Antisense 5 'to 3': AACGUGUCAUCAGCAUAGCgg) formed from the antisense sequence shown in siRAB3B # 2 A nucleotide sequence and a control siRNA (siNegaCont: Silencer (registered trademark) Select Negative Control No. 1 siRNA; manufactured by Thermo Fisher Scientific) were used. The following conditions were applied to the reverse transfection method. 1x10 5 cells / 5 nM each siRNA / 1 μL Lipofectamine RNAiMAX Transfection Reagent / 1 mL Opti-MEM I Reduced Serum medium (Thermo Fisher Scientific). The results are shown in FIGS. 5 (a), 5 (b), 6 and 7.
図5(a)、(b)に示すように、RAB3B遺伝子発現を、siRNA（siRAB3B#1,#2）を用いてノックダウンしたところ、RAB3Bが遺伝子レベル(移入3日後)でもタンパク質レベル(移入4日後)でも発現が抑制されていることが確認された。また、図6に示すように、SK-HEP-1株においてRAB3B遺伝子発現を、siRNA（siRAB3B#1,#2）を用いてノックダウンしたところ、浮遊細胞塊誘導条件下では「浮遊細胞塊」から「接着性の細胞」への転換が見られた。さらに、図7に示すように、コントロール(siNegaCont)と比較してsiRNAによるRAB3Bノックダウン株（siRAB3B#1,#2）では接着細胞数／浮遊細胞塊細胞数比が2倍以上上昇していた。このことから、RAB3B遺伝子の発現を抑制することで、癌幹細胞における接着性(adherent)誘導、換言すれば、幹細胞性（stemness）解除を誘導することができることが明らかとなった。なお、細胞における接着性が誘導されれば、血中で浮遊状態での生存がしづらく、転移能が低下すると考えられている。 As shown in FIGS. 5 (a) and (b), when RAB3B gene expression was knocked down using siRNA (siRAB3B # 1, # 2), RAB3B was expressed at the gene level (3 days after transfer) and at the protein level (transferred 3 days after transfer). 4 days later), it was confirmed that the expression was suppressed. In addition, as shown in FIG. 6, when the RAB3B gene expression was knocked down in the SK-HEP-1 strain using siRNA (siRAB3B # 1, # 2), the "suspended cell mass" To "adherent cells". Furthermore, as shown in FIG. 7, the ratio of the number of adherent cells / the number of floating cell mass cells was more than doubled in the RAB3B knockdown strains (siRAB3B # 1, # 2) by siRNA compared to the control (siNegaCont). . From this, it became clear that suppressing the expression of the RAB3B gene can induce induction of adhesiveness (in other words, release of stemness) in cancer stem cells. It is considered that if the cell adhesion is induced, it is difficult to survive in a suspended state in blood and the metastatic ability is reduced.
［実施例５］
（SK-sphere特異的microRNAの探索）
microRNAは配列依存的に複数の遺伝子発現を制御するnon-coding RNAである。近年、microRNAは細胞内だけでなく、エクソソーム小胞に内包され細胞外に分泌されることも知られるようになった。すなわち、microRNAは細胞内における一つの遺伝子発現制御だけでなく、他の細胞における遺伝子発現をも制御しうる。まず、SK-HEP-1とSK-sphereにおける培地中のRNA濃度を調べたところ、SK-sphereはSK-HEP-1と比較してRNA濃度が上昇していた（図8(a)）。そのため、CSLCにおいてはmicroRNAによって癌微小環境や周辺細胞の制御が考えられた。
[Example 5]
(Search for SK-sphere specific microRNA)
MicroRNAs are non-coding RNAs that control the expression of multiple genes in a sequence-dependent manner. In recent years, it has become known that microRNAs are included not only in cells but also in exosome vesicles and secreted out of cells. That is, the microRNA can control not only one gene expression in a cell but also gene expression in another cell. First, when the RNA concentrations in the medium in SK-HEP-1 and SK-sphere were examined, the RNA concentration of SK-sphere was higher than that of SK-HEP-1 (FIG. 8 (a)). Therefore, it was considered that microRNA controls tumor microenvironment and surrounding cells in CSLC.
そこで、SK-sphere特異的microRNAの探索を行った。microRNAの発現解析は3D-Gene miRNA Labeling kit及び3D-Gene Human miRNA Oligo Chip（東レ社製）を用いて行った。サンプルとしてSK-HEP-1、SK-sphere及びSK-sphereの培養上清を用い、ハイブリダイゼーションシグナルの相対蛍光強度をそれぞれの中央値を指標として標準化した後に、それぞれにおけるmicroRNAの発現量を比較し、Log2 Ratio及びFisher比を求めた。その結果、SK-sphere特異的microRNAとしてhas-miR-15a-5p (MIMAT0000068)を同定した。SK-HEP-1、SK-sphere及びSK-sphereの培養上清それぞれの発現量の結果を図8(b)に示す。 Therefore, we searched for SK-sphere specific microRNA. The microRNA expression analysis was performed using a 3D-Gene miRNA Labeling kit and a 3D-Gene Human miRNA Oligo Chip (manufactured by Toray Industries, Inc.). Using the culture supernatants of SK-HEP-1, SK-sphere and SK-sphere as samples, the relative fluorescence intensity of the hybridization signal was normalized using the median value as an index, and the expression levels of microRNA in each were compared. , Log2 Ratio and Fisher ratio were determined. As a result, has-miR-15a-5p (MIMAT0000068) was identified as a SK-sphere-specific microRNA. FIG. 8 (b) shows the results of the expression levels of SK-HEP-1, SK-sphere, and the culture supernatant of SK-sphere.
図8(b)において、has-miR-15a-5pはSK-HEP-1と比較してSK-sphereにて発現が低下し、SK-sphere培養上清における発現割合も低いmicroRNAであることが明らかとなった。興味深いことに、has-miR-15a-5pはRAN3B mRNAを標的とするということは配列上からは予測されていないが、図8(c)に示すように、has-miR-15a-5のmimic RNA(mirVana miRNA mimic, Thermo Fisher Scientific社製, mature miRNA Sequence; UAGCAGCACAUAAUGGUUUGUG（配列番号7）, stem loop sequence; CCUUGGAGUAAAGUAGCAGC ACAUAAUGGUUUGUGGAUUU UGAAAAGGUGCAGGCCAUAU UGUGCUGCCUCAAAAAUACA AGG（配列番号8)の移入によってRAB3BのmRNA発現抑制が観察された。has-miR-15a-5pのmimic RNAの移入は以下の条件下でのReverse transfection法により行った；1x105 cells/ 10nM each RNA/ 1μL Lipofectamine RNAiMAX Transfection Reagent/ 1mL Opti-MEM I Reduced Serum培地 (Thermo Fisher Scientific社製)。コントロールRNA (cont-RNA) としてmirVana miRNA Mimic, Negative Control No. 1 (Thermo Fisher Scientific社製)を同濃度用いた。 In FIG. 8 (b), has-miR-15a-5p is a microRNA whose expression is reduced in the SK-sphere compared to SK-HEP-1, and the expression ratio in the SK-sphere culture supernatant is also low. It became clear. Interestingly, it is not predicted from the sequence that has-miR-15a-5p targets RAN3B mRNA, but as shown in Figure 8 (c), the mimic of has-miR-15a-5 RNA (mirVana miRNA mimic, manufactured by Thermo Fisher Scientific, mature miRNA Sequence; UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO: 7), stem loop sequence; CCUUGGAGUAAAGUAGCAGC ACAUAAUGGUUUGUGGAUUU UGAAAAGGUGCAGGCCAUAU UGUGCUGCCUCAAAAAUAB AGG ABAG of AGG AB was detected by AGGAAB). Transfer of the mimic RNA of has-miR-15a-5p was performed by the reverse transfection method under the following conditions; 1 × 10 5 cells / 10 nM each RNA / 1 μL Lipofectamine RNAiMAX Transfection Reagent / 1 mL Opti-MEM I Reduced Serum medium (Thermo MirVana miRNA Mimic, Negative Control No. 1 (manufactured by Thermo Fisher Scientific) was used as the control RNA (cont-RNA) at the same concentration.
［実施例６］
（CRISPR/Cas9によるノックアウト解析）
CRISPR/Cas9を用いたゲノム編集技術を用いて、SK-HEP-1株を親株としてmono-allelic RAB3B knock-out株を樹立した。ゲノム編集にはCas9 mRNA及び配列番号9に示す(-)鎖配列（GTTTCACCCGCTTCTCGTGA）及び続くCGGをそれぞれ標的配列及びPAM配列とし、配列番号10に示す配列 (GUUUCACCCGCUUCUCGUGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu)をguide RNAとして合成し、guide RNAをlipofectamine RNAi Max（Thermo Fisher Scientific社製）によりSK-HEP-1細胞株に移入した後にsingle cell cloningを行った。得られたcloneからgenome DNAを抽出し、サンガーシーケンスにより変異導入を確認した。上記guide RNAとCas9 mRNAを用いてゲノム編集を行うことで、図9に示されるように、SK-HEP-1細胞株におけるgenome DNA中のPAM配列 (下線にて示したCCG) に隣接する標的配列に二本鎖切断が起こり、結果として配列番号１に示されるRAB3Bのアミノ酸配列が、配列番号11に示されるアミノ酸配列へと変異したクローンが得られた。図9右側上段はRAB3B野生型配列を示し、下段には変異クローンにおける配列を示した。野生型の全長219アミノ酸に対して変異型では67番目のアミノ酸以降の配列が異なり、全長も102アミノ酸となっている。左側は塩基配列を示し、guide RNAは相補鎖に対して設計し、下段に示されるようにcoding領域の200番目のアデニン（A）と201番目のシトシン（C）との間にAが挿入された配列が変異クローンにおけるRAB3B配列である。
[Example 6]
(Knockout analysis by CRISPR / Cas9)
Using a genome editing technology using CRISPR / Cas9, a mono-allelic RAB3B knock-out strain was established using the SK-HEP-1 strain as a parent strain. For genome editing, Cas9 mRNA and the (-) chain sequence shown in SEQ ID NO: 9 (GTTTCACCCGCTTCTCGTGA) and the subsequent CGG as the target sequence and PAM sequence, respectively, and the sequence shown in SEQ ID NO: 10 Was transferred to SK-HEP-1 cell line using lipofectamine RNAi Max (manufactured by Thermo Fisher Scientific), and then single cell cloning was performed. Genome DNA was extracted from the obtained clone, and mutation introduction was confirmed by Sanger sequencing. By performing genome editing using the guide RNA and Cas9 mRNA, as shown in FIG. 9, the target adjacent to the PAM sequence in the genome DNA (CCG underlined) in the SK-HEP-1 cell line A double-strand break occurred in the sequence, resulting in a clone in which the amino acid sequence of RAB3B shown in SEQ ID NO: 1 was mutated to the amino acid sequence shown in SEQ ID NO: 11. The upper part on the right side of FIG. 9 shows the RAB3B wild-type sequence, and the lower part shows the sequence in the mutant clone. Compared to the wild-type total length of 219 amino acids, the mutant type differs in the sequence after the 67th amino acid, and has a total length of 102 amino acids. The left side shows the nucleotide sequence. The guide RNA is designed for the complementary strand, and A is inserted between the adenine (A) at position 200 and the cytosine (C) at position 201 in the coding region, as shown at the bottom. Is the RAB3B sequence in the mutant clone.
HuH-7細胞株、上記で樹立したmono-allelic RAB3B knock-out細胞株(RAB3B-KO8細胞株)及びSK-HEP-1細胞株それぞれをDMEM培地又はNSC培地で培養し、RAB3B発現を確認した結果を図10に示す。図10(a)は上記それぞれの細胞株においてRNA-seq解析からのRAB3B mRNA発現を示す。RAB3B-KO8細胞株ではsphere誘導のNSC培地においてもSK-HEP-1よりも低いRAB3Bの発現レベルであった。また、図10(b)は上記それぞれの細胞株においてウエスタンブロット解析からのRAB3Bタンパク発現及び内部標準としてvalosin containing protein (VCP)発現を示す。normal培地とsphere誘導のNSC培地の両方において、SK-HEP-1と比較してRAB3B-KO8細胞株におけるRAB3B発現低下を認めた。 HuH-7 cell line, mono-allelic RAB3B knock-out cell line established above (RAB3B-KO8 cell line) and SK-HEP-1 cell line were cultured in DMEM medium or NSC medium, respectively, to confirm RAB3B expression. The results are shown in FIG. FIG. 10 (a) shows RAB3B mRNA expression from RNA-seq analysis in each of the above cell lines. In the RAB3B-KO8 cell line, the expression level of RAB3B was lower than that of SK-HEP-1 even in the sphere-induced NSC medium. FIG. 10 (b) shows the expression of RAB3B protein from Western blot analysis and the expression of valosin containing protein (VCP) as an internal standard in each of the above cell lines. In both the normal medium and the sphere-induced NSC medium, RAB3B expression was reduced in the RAB3B-KO8 cell line compared to SK-HEP-1.
次に、SK-HEP-1細胞株及び上記で樹立したmono-allelic RAB3B knock-out (RAB3B-KO8)株の形態を調べた。結果を図11に示す。図11(a)はSK-HEP-1細胞株をDMEM培地で培養した場合、図11(b)はRAB3B-KO8細胞株をDMEM培地で培養した場合、図11(c)はSK-HEP-1細胞株をsphere誘導のNSC培地で培養した場合、図11(d)はRAB3B-KO8細胞株をsphere誘導のNSC培地で培養した場合である。図11(d)に示すように、RAB3B-KO8細胞株をsphere誘導のNSC培地で培養してもsphereの形成があまり生じておらずRAB3B-KO8細胞株ではsphere形成の著しい抑制が観察された。 Next, the morphology of the SK-HEP-1 cell line and the mono-allelic RAB3B knock-out (RAB3B-KO8) strain established above were examined. The results are shown in FIG. FIG. 11 (a) shows the case where the SK-HEP-1 cell line was cultured in the DMEM medium, FIG. 11 (b) shows the case where the RAB3B-KO8 cell line was cultured in the DMEM medium, and FIG. 11 (c) shows the case where the SK-HEP- FIG. 11 (d) shows the case where one cell line was cultured in a sphere-derived NSC medium, and FIG. 11 (d) shows the case where the RAB3B-KO8 cell line was cultured in a sphere-derived NSC medium. As shown in FIG. 11 (d), even when the RAB3B-KO8 cell line was cultured in a sphere-derived NSC medium, sphere formation was not much observed, and marked suppression of sphere formation was observed in the RAB3B-KO8 cell line. .
さらに、レスキュー実験として、RAB3B-KO8細胞株にpcDNA3.1(-) vector 、又はpRAB3B（pcDNA3.1(-) vectorにRAB3Bをクローニングしたもの）をlipofectamine 3000を用いて移入した後に、抗生物質G418により薬剤選択を行った。得られた、pcDNA3.1(-) vectorのみのmock移入株とpRAB3B移入株をそれぞれsphere誘導のNSC培地にて7-24日間のsphere誘導処置を行い、顕微鏡下にて観察した結果を図12に示す。図12に示すように、pRAB3B移入株におけるsphere形成能の回復が見られた。上記結果から、RAB3Bがsphere形成能に関与していることが確認された。 Furthermore, as a rescue experiment, pcDNA3.1 (−) vector or pRAB3B (RAB3B was cloned into pcDNA3.1 (−) vector) was transferred to RAB3B-KO8 cell line using lipofectamine 3000, and then antibiotic G418 was used. Was used for drug selection. The resulting mock-transferred strain of pcDNA3.1 (-) vector alone and the pRAB3B-transferred strain were each subjected to sphere-inducing treatment for 7 to 24 days in a sphere-inducing NSC medium, and observed under a microscope. Shown in As shown in FIG. 12, recovery of the sphere forming ability was observed in the pRAB3B-transferred strain. From the above results, it was confirmed that RAB3B was involved in the sphere forming ability.
［実施例７］
（免疫監視機構関連因子の解析）
上記で作製したSK-HEP-1細胞株（親株：parent）及びSK-sphere株(Sphere)における免疫監視機構に関連するmRNAの発現量を調べるためにRNAシーケンスを行い、さらにその発現量の比（sphere/parent）を調べた。結果を図13に示す。
[Example 7]
(Analysis of factors related to immune monitoring mechanism)
RNA sequencing was performed to examine the expression level of mRNA related to the immune surveillance mechanism in the SK-HEP-1 cell line (parent strain: parent) and SK-sphere strain (Sphere) prepared above, and the ratio of the expression levels was further determined. (Sphere / parent). FIG. 13 shows the results.
図13から明らかなように、PD-1のリガンドであるPD-L1及びPD-L2はSphere/parentとしてそれぞれ1.2倍、2.0倍も発現が上昇していた。さらに、HLA-A、HLA-B、HLA-CやCEACAM、MICAやMICB、ADAM17においても発現が亢進していた。このほか、図13には示していないが、NCR3LG1 (B7-H6)は発現が亢進(Log2=0.95)し、HMGB1は発現が減少(Log2=-0.75)し、CD276(B7-H3)、PVR (CD155)、及びCD47はSphere株と親株とで有意な差は見られなかった。VTCN1(B7-H4)、HHLA2 (B7-H5)、LGALS9 (galectin 9)、BTLA (CD272)、TIGIT、及びCD96については、有意な発現自体が見られなかった。 As is apparent from FIG. 13, the expression of PD-1 ligands PD-L1 and PD-L2 as Sphere / parent was increased by 1.2 times and 2.0 times, respectively. Furthermore, expression was also enhanced in HLA-A, HLA-B, HLA-C, CEACAM, MICA, MICB, and ADAM17. In addition, although not shown in FIG. 13, NCR3LG1 (B7-H6) is upregulated (Log2 = 0.95), HMGB1 is downregulated (Log2 = -0.75), CD276 (B7-H3), PVR (CD155) and CD47 showed no significant difference between the Sphere strain and the parent strain. VTCN1 (B7-H4), HHLA2 (B7-H5), LGALS9 (galectin 9), BTLA (CD272), TIGIT, and CD96 did not show significant expression itself.
上記結果により、SK-HEP-1細胞株に対してSK-sphere株においてPD-L1、PD-L2及びHLA-ABCのmRNAの発現が上昇していたことから、さらにフローサイトメトリーによってPD-L1、PD-L2及びHLA-ABCタンパク質発現を解析した。結果を図14に示す。 According to the above results, the expression of mRNA of PD-L1, PD-L2 and HLA-ABC was increased in the SK-sphere strain relative to the SK-HEP-1 cell line, and the PD-L1 was further analyzed by flow cytometry. , PD-L2 and HLA-ABC protein expression were analyzed. The results are shown in FIG.
図14ら明らかなように、フローサイトメトリーによっても、sphere形成によりPD-L1及びPD-L2の発現量が増加することが確認された。 As is apparent from FIG. 14, it was confirmed by flow cytometry that the expression levels of PD-L1 and PD-L2 were increased by sphere formation.
［実施例８］
次に、SK-HEP-1細胞株及びSK-sphere株それぞれにおける膜結合MICA/B及び可溶性MICA/Bを調べた。膜結合MICAは抗MICA/B抗体を用いたフローサイトメトリーにより解析し、可溶性MICAは抗MICA抗体を用いたELISAにより解析した。結果を図15に示す。図15(a)により、sphereが形成されると膜結合型MICAの発現が低下し、他方、図15(b)により可溶性MICAの発現量が増加していた。免疫活性化を担うNKG2Dに対するリガンドの膜発現は活性化シグナルを引き起こし、一方、遊離したNKG2DリガンドはNKG2D活性化シグナルを抑制することが知られている。かかる結果より、膜結合型リガンドの減少によりNKG2Dからの活性化シグナルを減弱させると共に、分泌型リガンドの増加によっても活性化シグナルを抑制することで、CSLCが免疫逃避していることが明らかとなった。
Example 8
Next, membrane-bound MICA / B and soluble MICA / B in the SK-HEP-1 cell line and the SK-sphere line, respectively, were examined. Membrane-bound MICA was analyzed by flow cytometry using an anti-MICA / B antibody, and soluble MICA was analyzed by ELISA using an anti-MICA antibody. The results are shown in FIG. As shown in FIG. 15 (a), when the sphere was formed, the expression of membrane-bound MICA was reduced, while in FIG. 15 (b), the expression level of soluble MICA was increased. It is known that membrane expression of a ligand for NKG2D responsible for immune activation causes an activation signal, whereas released NKG2D ligand suppresses the NKG2D activation signal. From these results, it was revealed that CSLC is immune evading by reducing the activation signal from NKG2D by decreasing the membrane-bound ligand and suppressing the activation signal by increasing secreted ligand. Was.
［実施例９］
（RAB3Bのノックアウトによる免疫監視機構関連因子の発現への影響）
上記結果に基づいて、RAB3Bをノックアウトした場合の免疫逃避機構関連因子の発現について確認するために、SK-HEP-1細胞株をDMEM培地で培養したSK-HEP-1/DMEM、SK-HEP-1細胞株をNSC培地で培養したSK-sphere/NSC、RAB3B-KO8をDMEMで培養したRAB3B-KO8/DMEM、及びRAB3B-KO8をNSCで培養したRAB3B-KO8/NSCそれぞれにおけるPD-L1、PD-L2、HLA-ABC、及びULBP1の発現をフローサイトメトリーで調べた。PD-L1の発現を調べた結果を図16に、PD-L2の発現を調べた結果を図17に、HLA-ABCの発現を調べた結果を図18に、ULBP1の発現を調べた結果を図19に示す。
[Example 9]
(Effect of knockout of RAB3B on expression of factors related to immune surveillance mechanism)
Based on the above results, in order to confirm the expression of immune escape mechanism-related factors when RAB3B is knocked out, SK-HEP-1 / DMEM, SK-HEP- 1 cell line cultured in NSC medium SK-sphere / NSC, RAB3B-KO8 cultured in DMEM RAB3B-KO8 / DMEM, and RAB3B-KO8 cultured in NSC RAB3B-KO8 / NSC PD-L1, PD in each -The expression of L2, HLA-ABC and ULBP1 was examined by flow cytometry. The results of examining the expression of PD-L1 are shown in FIG. 16, the results of examining the expression of PD-L2 are shown in FIG. 17, the results of examining the expression of HLA-ABC are shown in FIG. 18, and the results of examining the expression of ULBP1 are shown in FIG. As shown in FIG.
図16から明らかなように、SK-HEP-1細胞株をNSC培地培養すると、DMEM培地で培養した場合と比較してPD-L1陽性細胞の割合が増加しているのに対し、RAB3B-KO8株をNSC培地培養すると、DMEM培地で培養した場合と比較してPD-L1陽性細胞の割合が減少していた。また、図17から明らかなように、SK-HEP-1細胞株をNSC培地培養すると、DMEM培地で培養した場合と比較してPD-L2陽性細胞の割合が増加しているのに対し、RAB3B-KO8株をNSC培地培養すると、DMEM培地で培養した場合と比較してPD-L2陽性細胞の増加の割合が減少していた。一方、図18、19から明らかなように、HLA-ABCやULBP1においてはRAB3Bをノックアウトすることによる発現の変化はみられなかった。かかる結果より、RAB3Bをノックアウトすることで、癌細胞におけるPD-L1及びPD-L2の発現を抑制可能であることが確認された。換言すれば、RAB3Bの発現を抑制すれば、癌の免疫逃避機構を変化させてT細胞の活性化抑制を解除できることが明らかとなった。 As is clear from FIG. 16, when the SK-HEP-1 cell line was cultured in the NSC medium, the percentage of PD-L1 positive cells was increased as compared to the case where the SK-HEP-1 cell line was cultured in the DMEM medium, whereas RAB3B-KO8 When the strain was cultured in NSC medium, the proportion of PD-L1-positive cells was reduced as compared to when the strain was cultured in DMEM medium. As is clear from FIG. 17, when the SK-HEP-1 cell line was cultured in the NSC medium, the ratio of PD-L2 positive cells was increased as compared to the case where the SK-HEP-1 cell line was cultured in the DMEM medium, whereas RAB3B When the -KO8 strain was cultured in the NSC medium, the rate of increase in the number of PD-L2-positive cells was decreased as compared to the case where the -KO8 strain was cultured in the DMEM medium. On the other hand, as is clear from FIGS. 18 and 19, in HLA-ABC and ULBP1, no change in expression due to knockout of RAB3B was observed. From these results, it was confirmed that knockout of RAB3B can suppress the expression of PD-L1 and PD-L2 in cancer cells. In other words, it was revealed that suppressing the expression of RAB3B can release the suppression of T cell activation by changing the immune escape mechanism of cancer.
Claims (7)
（I）RAB3B遺伝子を標的とし、RAB3B遺伝子の発現を抑制する核酸分子；
（II）配列番号1に示されるRAB3Bタンパク質に結合する低分子化合物； An agent for suppressing the expression of an immune checkpoint factor in cancer cells, comprising any one of the following (I) or (II) as an active ingredient:
(I) a nucleic acid molecule that targets the RAB3B gene and suppresses the expression of the RAB3B gene;
(II) a low-molecular compound that binds to the RAB3B protein represented by SEQ ID NO: 1;
interfering RNA(siRNA)、micro RNA、small hairpin RNA(shRNA)、guide RNA(gRNA)、アンチセンス核酸、又はリボザイムからなる群から選択される少なくとも１種の核酸分子であることを特徴とする請求項１〜４のいずれか記載の癌細胞における免疫チェックポイント因子の発現抑制剤。 The nucleic acid molecule is small
Claims are at least one nucleic acid molecule selected from the group consisting of interfering RNA (siRNA), micro RNA, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, or ribozyme. 5. The agent for suppressing the expression of an immune checkpoint factor in cancer cells according to any one of 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018173611A JP7231917B2 (en) | 2018-09-18 | 2018-09-18 | Immune checkpoint factor expression inhibitor in cancer cells and pharmaceutical composition for cancer treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018173611A JP7231917B2 (en) | 2018-09-18 | 2018-09-18 | Immune checkpoint factor expression inhibitor in cancer cells and pharmaceutical composition for cancer treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020045303A true JP2020045303A (en) | 2020-03-26 |
| JP7231917B2 JP7231917B2 (en) | 2023-03-02 |
Family
ID=69899335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018173611A Active JP7231917B2 (en) | 2018-09-18 | 2018-09-18 | Immune checkpoint factor expression inhibitor in cancer cells and pharmaceutical composition for cancer treatment |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7231917B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023219119A1 (en) * | 2022-05-12 | 2023-11-16 | 国立大学法人 東京大学 | Method for producing extracellular vesicles containing bioactive substance |
-
2018
- 2018-09-18 JP JP2018173611A patent/JP7231917B2/en active Active
Non-Patent Citations (2)
| Title |
|---|
| TAN, P.Y. ET AL., MOL CELL BIOL, vol. 32, no. 2, JPN6022030426, 2012, pages 399 - 414, ISSN: 0004833633 * |
| YE, F. ET AL., J TRANSL MED, vol. 12, no. 17, JPN6022030424, 2014, pages 1 - 10, ISSN: 0004833632 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023219119A1 (en) * | 2022-05-12 | 2023-11-16 | 国立大学法人 東京大学 | Method for producing extracellular vesicles containing bioactive substance |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7231917B2 (en) | 2023-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5931897B2 (en) | Materials and methods associated with microRNA-21, mismatch repair and colorectal cancer | |
| EP3420086A1 (en) | COMPOSITIONS AND METHODS OF USING piRNAS IN CANCER DIAGNOSTICS AND THERAPEUTICS | |
| Holen et al. | Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo | |
| US20220110963A1 (en) | Reverse transcriptase blocking agents and methods of using the same | |
| WO2016038550A1 (en) | Inhibition of prmt5 to treat mtap-deficiency-related diseases | |
| JP2016064989A (en) | Pharmaceutical composition for treating cancer, and method for evaluating sensitivity to treatment by pd-1 inhibitor | |
| JPWO2019093502A1 (en) | Cancer promoter expression inhibitor, screening method for its active ingredient, expression cassette, diagnostic agent, and diagnostic method useful for the method. | |
| JP5933010B2 (en) | Cancer treatment | |
| WO2016089928A1 (en) | Methods for treating and assessing tumor invasion and metastasis | |
| JP7231917B2 (en) | Immune checkpoint factor expression inhibitor in cancer cells and pharmaceutical composition for cancer treatment | |
| US8921333B2 (en) | Therapeutic agent for tumor | |
| EP2810655A1 (en) | Novel antitumor agent and method for screening same | |
| CA2932910A1 (en) | Methods for diagnosing and treating metastatic cancer | |
| US20210277395A1 (en) | Methods and compositions for modulating lncrnas and methods of treatment based on lncrna expression | |
| US20200140861A1 (en) | Long non-coding rna in cancer | |
| JP7226763B2 (en) | Agent for reducing drug resistance in cancer stem cells, inhibitor for metastatic potential in cancer stem cells, and method for predicting risk of metastatic recurrence of cancer | |
| JP2021514195A (en) | Targeted cancer treatment using mutant P53-specific siRNA | |
| WO2010050328A1 (en) | Tumor metastasis inhibitor | |
| JP5098013B2 (en) | Method for establishing cell line of human malignant ovarian germ cell tumor, human malignant ovarian embryo tumor cell line, and use thereof | |
| WO2022238542A2 (en) | Krab-containing zinc finger protein and cancer | |
| WO2023164689A2 (en) | Targeting neuropilin 2 (nrp2) in lethal prostate cancer | |
| Miglio et al. | The silencing of the L1-MET chimeric transcript activates cancer cell death program and inhibits the expression of crucial oncoproteins in lung cancer cells | |
| WO2021173952A1 (en) | Compositions and methods for detecting bcl2l14 and etv6 gene fusions for determining increased drug resistance | |
| KR20230004458A (en) | Methods and compositions for the treatment of APC-deficient cancers | |
| WO2022015532A1 (en) | Compositions and methods for detecting gene fusions of rad51ap1 and dyrk4 and for diagnosing and treating cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210712 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220726 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20220915 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221118 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230207 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230210 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7231917 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |